Cells have a variety of fail-safe mechanisms, one of which is to arrest the cell division of damaged chromosomal DNA and to repair the damage, thus preventing mutations from settling. When chromosomal DNA damaged by UV and the like is continued to undergo cell division in a condition where the damage is not repaired, the damaged chromosomal DNA will be replicated so as to accumulate mutations. This leads to an increase in the incidence of cancer cells. Accordingly, when DNA is damaged, cells operate a process of repairing the damage and an intracellular feedback mechanism of arresting the cell division until the repair of DNA damage is over, followed by inhibiting the development of cancers. Such a feedback mechanism is mediated by checkpoints in each cycle of cell division. The overall function of these checkpoints is to detect damaged or abnormally structured DNA and to coordinate cell-cycle progression with DNA repair (Robert T. Genes & development, 15:2177-2196, 2001). Typically, cell-cycle checkpoint activation slows or arrests cell-cycle progression, thereby allowing time for appropriate repair mechanisms to correct genetic lesions before they are passed on to the next generation of daughter cells. In certain cells, such as thymocytes, checkpoint proteins link DNA strand breaks to apoptotic cell death via the induction of p53 (Robert T. Genes & development, 15:2177-2196, 2001).
Cell-cycle checkpoints which are initiated by DNA damages are mainly regulated by ATM (ataxia-telangiectasia-mutated) and ATR (ATM- and Rad3-ralated) proteins (Shiloh, Y. Curr. Opin. Gent. Dev., 11:71-77, 2001; Abraham, R. T. Genes Dev., 15:2177-2196, 2001). Such proteins play a key role in the early signal transduction via the cell-cycle checkpoints. ATM- and ATR-deficient cells showed defects in arresting the cell cycle in response to radiation. Particularly, the ATM-deficient cells showed serious defects in G1, S and G2 checkpoints (Robert T. Genes & development, 15:2177-2196, 2001), and serious damages called “double strand breaks” occurred in the ATR -deficient cells. Furthermore, it was known that the incidence of tumor is greatly increased by the mutation of ATM/ATR.
ATM and ATR are highly homologous to each other and use the same substrate. However, they are different in that their activities are increased by different genotoxic stresses. ATM responds to agents, such as IR (ionizing radiation) that breaks double strands DNA, whereas ATR responds to agents (including IR) that cause bulky adducts on DNA or single strand DNA. Furthermore, ATM and ATR are activated by different methods. ATM activation requires autophosphorylation that results in the disruption of an ATM dimer (Bakkenist, C. J. et al., Nature, 421:499-506, 2003). How autophosphorylation of ATM triggered is still unknown. ATR may also be autophosphorylated, but it is not evident that ATR forms either an inactive dimer or an active monomer in cells. Also, it is not yet clear that other subunits or cofactors are required for the activation of ATM/ATR. In addition, the intracellular biochemical mechanism of a signal transduction system where the DNA damage causes the activation and operation of ATM/ATR was not completely established.
Target proteins known to be phosphorylated directly by ATM/ATR include p53, chk1, chk2, c-Abl, RPA and the like, of which p53 is phosphorylated on serine 15 by ATM/ATR. It was reported that the over-expression of p53 arrests G2 and suppresses the synthesis of two proteins, CDK1 (cyclin-dependent kinase 1) and cyclin BI, which are required for the entry of cells from G2 to M. Thus, p53 does not only the function of inhibiting the abnormal division and proliferation of cells, but also the function of arresting the cell cycle so as to repair the damaged DNA when DNA was damaged. Recently, the mutation and loss of p53 genes are recognized as one of the most frequent genetic mutations, which is found not only in any certain cancer but in almost all types of cancer in human. Moreover, p53 activates the transcription of p21, another tumor suppressor gene, thereby inhibiting the G1/S transition and causing the p53-dependent apoptosis. p21 which is expressed by p53 was known to be a kind of a CKI (cyclin -dependent kinase inhibitor) which functions to inhibit the division and proliferation of cells. Accordingly, efforts for developing new anticancer agents using cell-cycle regulation factors or substances of activating the factors are now continued.
Meanwhile, aminoacyl-tRNA synthetases (ARSs) which are important enzymes catalyzing the first step in protein synthesis are multifunctional proteins involved in various biological functions (Ko et al., Proteomics, 2:1304-1310, 2002). Among them, various mammalian tRNA synthetases, such as MRS (methionyl-tRNA synthetase), QRS (glutaminyl-tRNA synthetase), RRS (arginyl-tRNA synthetase), KRS (Lysyl-tRNA synthetase), DRS (aspartyl-tRNA synthetase) and so on, bind to three non-enzyme cofactors, designated as p43, p38 and p18, to form a macromolecular protein complex (Han et al., Biochem. Biophys. Res. Commun., 303:985-993, 2003). Since ARSs are enzymes necessary for protein synthesis, this complex deems to be formed in order to facilitate protein synthesis.
Among the non-enzyme cofactors binding to ARSs, p43 is known to play an important role as a cytokine in immune response and angiogenesis (Ko et al., J. Biol. Chem., 276:23028-32303, 2001b; Park et al., J. Biol. Chem., 277:45234-45248, 2002). Furthermore, p38 was found to downregulate c-myc, a protoocogene, and to be involved in lung differentiation (Kim et al., Nat. Genet., 34:330-336, 2003). The last cofactor, p18, shows sequence homology to elongation factor subunits (EF-1) (Quevillon and Mirande, FEBS Lett., 395:63-67, 1996). Given this, p18 is presumed to be involved in protein synthesis. However, the biological functions of p18 are not yet clearly understood, and particularly, there is no study on the relation between p18 and cancer.